LILRB4 on multiple myeloma cells promotes bone lesion by p-SHP2/NF-κB/RELT signal pathway

Background Leukocyte Ig-like receptor B family 4 (LILRB4) as an immune checkpoint on myeloid cells is a potential target for tumor therapy. Extensive osteolytic bone lesion is the most characteristic feature of multiple myeloma. It is unclear whether ectopic LILRB4 on multiple myeloma regulates bone lesion. Methods The conditioned medium (CM) from LILRB4-WT and -KO cells was used to analyze the effects of LILRB4 on osteoclasts and osteoblasts. Xenograft, syngeneic and patient derived xenograft models were constructed, and micro-CT, H&E staining were used to observe the bone lesion. RNA-seq, cytokine array, qPCR, the activity of luciferase, Co-IP and western blotting were used to clarify the mechanism by which LILRB4 mediated bone damage in multiple myeloma. Results We comprehensively analyzed the expression of LILRB4 in various tumor tissue arrays, and found that LILRB4 was highly expressed in multiple myeloma samples. The patient’s imaging data showed that the higher the expression level of LILRB4, the more serious the bone lesion in patients with multiple myeloma. The conditioned medium from LILRB4-WT not -KO cells could significantly promote the differentiation and maturation of osteoclasts. Xenograft, syngeneic and patient derived xenograft models furtherly confirmed that LILRB4 could mediate bone lesion of multiple myeloma. Next, cytokine array was performed to identify the differentially expressed cytokines, and RELT was identified and regulated by LILRB4. The overexpression or exogenous RELT could regenerate the bone damage in LILRB4-KO cells in vitro and in vivo. The deletion of LILRB4, anti-LILRB4 alone or in combination with bortezomib could significantly delay the progression of bone lesion of multiple myeloma. Conclusions Our findings indicated that LILRB4 promoted the bone lesion by promoting the differentiation and mature of osteoclasts through secreting RELT, and blocking LILRB4 singling pathway could inhibit the bone lesion. Supplementary Information The online version contains supplementary material available at 10.1186/s13046-024-03110-y.


Introduction
Multiple myeloma is incurable and characterized by abnormal clonal proliferation of malignant plasma cells, and ranks second after non-Hodgkin's lymphoma in hematological malignancies [1].Extensive osteolytic bone lesion is the most characteristic feature of multiple myeloma, including bone pain, osteoporosis, pathological fractures, spinal cord compression and hypercalcemia [2].The balance between bone resorption and bone formation is maintained by osteoblasts, osteoclasts, osteocytes, bone marrow stromal cells (BMSCs) and immune cells to keep physiological bone remodeling [3], but multiple myeloma cells disrupt this balance by cell to cell dependent or independent manner to result in osteolytic bone lesions [4][5][6].VLA-4 on the surface of multiple myeloma binding to the VCAM-1 in BMSCs accelerated the homing of multiple myeloma cells and modified the bone marrow microenvironment [7,8].Multiple myeloma cells secreted RNAKL and interleukin-6 (IL-6), endocytosed osteoprotegerin (OPG) to result in the elevated ratio of RNAKL/OPG, which promoted the differentiation and maturation of osteoclast precursors [9][10][11][12][13].The drugs targeting RNAKL [14][15][16], syndecan-1 [17,18], Sclerostin [19], DKK-1 [20] and RUNX2 [21] have enrolled preclinical or clinical trials.In additional, the combination of bortezomib and daratumumab [22], or thalidomide and bortezomib resulted in significantly longer progression-free survival [23].Following autologous stem cell transplant (ASCT) significantly prolonged progression-free survival [24], and anti-BCMA CAR-T cells improved survival time in relapses or refractory multiple myeloma [25].Although great achievements have been made in the treatment of multiple myeloma, discovery of new molecules and explore their role in bone lesions will help to develop new drugs for multiple myeloma.
As a monocytic checkpoint, leukocyte Ig-like receptor B family 4 (LILRB4) is mainly expressed on the monocytic lineage including dendritic cells, monocytes, macrophages and osteoclast [26,27].LILRB4 on antigen presenting cells (APC) induced CD4 + Th (T helper) cell anergy, and the proliferation of CD8 + T suppressor cells (Ts) [28][29][30].Our previous study reported that apoE activated LILRB4 signaling to mediate immune escape and extramedullary infiltration of acute myeloid leukemia (AML) by cell-cell independent manner [31,32].LILRB4 is also expressed on tumor-infiltrating myeloid-derived suppressor cells (MDSCs) and tumorassociated macrophage to promote tumor progression [35][36][37][38].Fibronectin or galectin-8 binding to LILRB4 could enhance the suppressive phenotype of tumor-associated myeloid cells [33,34].The humanized monoclonal antibody, chimeric antigen receptor T (CAR-T) cells and the antibody drug conjugates (ADC) that specifically targeted LILRB4 could significantly inhibit the progression of AML [39][40][41], therefore, LILRB4 has been as a potential target for tumor therapy [38].In addition, LILRB4 has been reported to be expressed on non-small cell lung cancer to promote angiogenesis, invasion and metastasis [42], on osteoclast to inhibit differentiation and maturation of osteoclast [43], on several B lineage cells including plasma cells and plasmablasts (in particular those from lupus patients) [44], MLL-rearranged B-ALL, and on B-cell chronic lymphocytic leukemia (CLL) cells to control tumor progression [45].Taken together, LILRB4 plays a key role in tumorigenesis and immune escape.
Concordant with its expression in certain B lineage cells, it was reported that LILRB4 is expressed in myeloma cells [40,46].However, it is unknown whether LILRB4 is functional in myeloma pathogenesis.In this study, we used immunohistochemistry to comprehensively analyze the expression of LILRB4 in various tumor tissue arrays, and found that LILRB4 was highly expressed in multiple myeloma samples.This result was furtherly verified in fresh multiple myeloma samples by flow cytometry, which suggested that LILRB4 was ectopic expressed on the surface of malignant plasma cells.It was reported that ectopic LILRB4 in B-cell chronic lymphocytic leukemia was involved in the infiltration of lymphoid tissues [47], controlled the progression by suppressing the Akt pathway [45], but the role of LILRB4 in multiple myeloma has not been reported.Here, we found that LILRB4 was significantly positively correlated with bone injury in multiple myeloma.LILRB4 positive rather than negative cells significantly promoted the bone lesion in xenograft, syngeneic and patient derived xenograft models.Mechanistically, fibronectin or apoE binding to LILRB4 recruited p-SHP2 to activate NF-κB signal pathway, then promoted multiple myeloma cells to secret RELT.The overexpression of RELT in LILRB4-KO cells significantly promoted bone lesion.The deletion of LILRB4 or anti-LILRB4 combined with bortezomib (BTZ) could significantly delay the progression of bone lesion of multiple myeloma.Our data revealed a new function of LILRB4, elucidated a new mechanism by which multiple myeloma promotes bone damage.

Patient sample and flow cytometry
Multiple myeloma bone marrow specimens were collected from UTSW Medical Center, Yantaishan Hospital and Yuhuangding Hospital.The criteria for inclusion and exclusion of patients are as follows: newly diagnosed patients with multiple myeloma who have not received treatment.All patients provided written informed consent, and this study was approved by the Medical Ethics Committee of Binzhou Medical University (No: 2020-17).The patient's information was listed in Supplementary Table S1.

Immunohistochemistry
Immunohistochemistry was performed according to our previous reports [31,32].In brief, all tissue arrays were deparaffinized, hydrated, antigen retrieved, blocked, incubated with primary antibodies, dehydrated and mounted.Rabbit anti-human LILRB4 antibody (Clone No: 128-3) was used to staining [39].This antibody was a non-humanized monoclonal antibody derived from rabbit and the dilution of antibody was 1:200, and DAB exposure time was 90 secs.

The overexpression of LILRB4 in 5TGM1 and J558 cells
The human LILRB4 was overexpressed in 5TGM1 and J558 cells according to our previous reports [31,32], and the human LILRB4 and GFP double positive cells were sorted by BD FACSAria III, and named 5TGM1-vector, 5TGM1-LILRB4, J558-vector and J558-LILRB4.
For primary mouse cell co-culture, bone marrow derived mononuclear cells from C57BL/6 mice at the age of 6-8 weeks were cultured in 96-well plates in α-MEM supplemented with 10% FBS, 25 ng/ml M-CSF, and 100 ng/ml RANKL in the presence of J558-vector or -LILRB4 conditioned medium for 6 days.The cells were fixed in formalin and stained for TRAP using a TRAP staining kit according to the instructions.TRAP + cells containing 3 or more nuclei were counted as osteoclasts.

The overexpression of RELT in KMS26-LILRB4-KO cells
The RNA was extracted from KMS26-LILRB4-WT cells using RNAiso Plus reagent (CAT#:9108, TAKARA) and reverse-transcribed using PrimeScript ™ II 1st Strand cDNA Synthesis Kit (CAT#:6210A, TAKARA) according to the manufacturer's manual.The specific primer of RELT was as follows, and underline indicated the infusion sequence.
F: ATT TCC GGT GAA TTC ATG AAG CCA AGT CTG CTG TGC CGG C; R: CGC TCT AGA ACT AGT TCA GAT GAC CAG GTT GCT CTC ACT T The cDNA of RELT was cloned into the lentivirus vector by in-fusion, and correct by sequencing.The human RELT expression plasmids were overexpressed in KMS26-LILRB4-KO cells.The RELT positive cells named KMS26-LILRB4-KO-RELT were sorted by BD FACSAria III, and KMS26-LILRB4-KO-vector cell as control.

Cytokine antibody arrays
Cytokine Antibody Arrays (RayBio ® Human Angiogenesis Antibody Array G-Series 1000, CAT #: AAH-ANG-G1000-4, RayBiotech, Inc.) were used to detect the levels of cytokine in the conditioned medium as described in manufacturer's protocol.In a short, 100 μl 1 × blocking buffer was added into each well and incubated at room temperature for 30 min to block slides, then aspirated from each well.Next, 100 μl sample was added and incubated at room temperature for 2 hours.After washing, 70 μl 1 × biotin-conjugated anti-Cytokines were added to each well, and incubated at room temperature for 2 hours with gentle rocking.Washing steps were repeated, followed by addition of 70 μl of 1 × Streptavidin-Fluor to each sub-array to incubate at room temperature for 2 hours in dark room with gentle rocking.Streptavidin-Fluor reagent were aspirated from each well carefully before washing.The signals of cytokine array were scanned with a microarray scanner (InnoScan 300, Innopsys, France) at the appropriate wavelength (532 nm) for Cy3, and fluorescence intensity data were analyzed using Raybiotech Software (RayBiotech, Peachtree Corners, Georgia, USA).

RNA-sequencing
2 × 10 6 KMS26-LILRB4-WT, -KO and OPM2-LILRB4-WT, -KO cells were cultured and collected, and all cells (3 replicates for each cell) were mailed to BGI in dry ice.The RNA extraction, quality control and sequencing were completed by GBI, and all data were analyzed in BGI Dr. Tom system (https:// biosys.bgi.com/).

Real time PCR
Total RNA was extracted from cells using RNAiso Plus reagent (CAT#:9108, TAKARA) and reverse-transcribed using PrimeScript ™ II 1st Strand cDNA Synthesis Kit (CAT #:6210A, TAKARA) according to the manufacturer's manual.100 ng cDNA products were used for subsequent PCR analysis through ChamQ Universal SYBR qPCR Master Mix (CAT#: Q-711, Vazyme) on QuantStudio ™ Design & Analysis SE Software (Thermo Fisher Scientific).The specific primer for target genes were as follows. VEGF

Co-immunoprecipitation
KMS26-LILRB4-WT and -KO cells were cultured, collected and washed twice with ice PBS (pH 7.4).Then, the cells were re-suspended by IP lysis buffer with protease inhibitors cocktail (Cat#: 04693116001, Roche) and incubated on ice for 30 mins.The supernatant was collected by centrifugation (4°C, 14,000 g, 10 mins).The cell lysate (600 μg) was divided into two parts and incubated with IgG or 128-3 [39] (anti-LILRB4) overnight at 4°C on a shaker, respectively.Next, Protein G Magnetic Beads were added and incubated for 2 h at 4°C on a shaker.Protein G Magnetic beads-Ab-Ag complex was washed 3 times, and heated at 95°C for 5 min in 1 × loading buffer.The samples were separated by SDS-PAGE, and antiphospho-SHP2 (Y580, Cat#:5431, Cell signaling technology) was incubated overnight.

ELISA
The levels of RELT in patient serum and conditioned medium (CM) were measured using a RELT ELISA Kit (CAT#: SEK10530, Sino biological).Briefly, diluted captured antibodies were coated to a 96 well plate and incubated overnight at 4℃. Standards and samples were added into the wells, and RELT bound to the immobilized antibody.The wells were washed and a horseradish peroxidase conjugated rabbit anti-human RELT monoclonal antibody was added.The wells were washed and TMB substrate solution was loaded.To end the enzyme reaction, the stop solution was added and absorbances of the microwell were read at 450 nm.

Luciferase reporter assay
The core promoter of RELT was amplified from the genomic DNA of KMS26 cells, and cloned into pGL3basic luciferase reporter vector by in-fusion, and was correct by sequencing.The primer was as follows, and underline indicated the in-fusion sequence.
F: 5'-TAG CCC GGG CTC GAG TTT AGA ACC CCA CCC CCA GCCAT-3' R: 5'-CGG AAT GCC AAG GCTT ACA CGG GGA GGC AGG GGG CGCCA-3' The expression vector of LILRB4 was conserved by our lab [31,32], and the coding sequence (CDS) of p65 was amplified and cloned into pcDNA3.1(+) by in-fusion.The specific primer targeting p65 was as follows and the underline indicated the in-fusion sequence.The p65 construct was correct by sequencing.

Multiple myeloma mouse model by tail vein
A total of 1 × 10 6 KMS26-and OPM2-LILRB4-WT, -KO cells were collected and re-suspended in 200 µl ice PBS (pH = 7.4), and the cells were injected into NSG mice by tail vein.For the KMS26 mice model, tumor progression was monitored by bioluminescence imaging (IVIS Lumina, PerkinElmer).After 5 weeks, the mice were sacrificed under anesthesia.The mice were raised until death in order to plot the survival curves in OPM2 mice model.Flow cytometry was used to detect the GFP + cell percentage in peripheral blood.The skull and tibia were stripped and fixed in formalin, and the bone lesion was scanned by micro-CT (Quantum GX, PerkinElmer).

5TGM1 multiple myeloma mouse model
A total of 1 × 10 6 5TGM1-vector and -LILRB4 cells were cultured, collected and re-suspended in 200 µl ice PBS (pH = 7.4), then were injected into C57BL/6-Rag2 −/− homozygous mice by tail vein.Bioluminescence imaging (IVIS Lumina, PerkinElmer) was used to monitor the progression of multiple myeloma.After 35 days, the mice were sacrificed under anesthesia, and micro-CT was used to scan bone lesion.

Multiple myeloma mouse model by intratibial injection
KMS26-LILRB4-WT, KMS26-LILRB4-KO-vector and -RELT cells were prepared, and NSG mice (4-6 weeks, female) were anesthetized by isoflurane.The tibial plateau was shaved and sterilized with 75% medical ethanol.At 5 mm below the knee joint, 3 mm incision fully exposed the tibia plateau, and 1 ml syringe was used to make a hole.The cells (1 × 10 6 in 10 µl micro-injector) were slowly injected into bone cavity, and the incision was sterilized.After 7 days, micro-CT was used to scan bone lesion.

PDX model of multiple myeloma
The bone marrow of newly diagnosed patients with multiple myeloma was provided in sterile anticoagulant tube by Yantaishan and Yuhuangding Hospital.CD45 − CD38 + LILRB4 − and CD45 − CD38 + LILRB4 + cells were sorted by BD FACSAria III.The same amount of cells were injected into NSG mice (4-6 weeks, female) by intratibial injection.When mice died naturally, the bone lesion was observed by micro-CT.

Multiple myeloma mouse model was treated by BTZ
A total of 1 × 10 6 KMS26-LILRB4-WT and -KO cells were injected into NSG mice (4-6 weeks, female) by tail vein.After 7 days, BTZ (1.0 mg/kg) or PBS were injected into NSG mice by intraperitoneal injection and twice each week.The mice were sacrificed under anesthesia on the 33 rd day, and the bone lesion was evaluated by micro-CT.

Anti-LILRB4 alone or in combination with BTZ was used to treat multiple myeloma mouse model
Anti-LILRB4 was produced according to our previous report [39].A total of 1 × 10 6 KMS26-LILRB4-WT cells were injected into NSG mice by tail vein.Isotype (CAT#: A2051, Selleck) (200 μg/each mouse), anti-LILRB4 (200 μg/each mouse) or BTZ (1.0 mg/kg) were used to treat the mouse model every 3 days from the 2 nd day.After 40 days, the mice were sacrificed, and bone was scanned by micro-CT.

Histology
The tibia was fixed with paraformaldehyde, decalcified with 10% EDTA for two weeks, embedded in paraffin, and sectioned along the mid-sagittal plane in 4.5 mmthick sections.Hematoxylin and eosin (H&E) staining was performed to observe bone morphology.

Micro-CT analysis
The formalin on the bone sample was cleaned, and the bone was scanned using a small animal in vivo micro-CT imaging system (Quantum GX, PerkinElmer) with 4.5 μm voxel size for 14 min, X-ray source set to 80000 mGy, 90 kV and 88μA.To observe cortical bone lesions, the entire femur image was imported into the software for 3D reconstruction using the same threshold.For trabecular microstructure, 100 slices, corresponding to a 1 mm region underneath the growth plate, were obtained with 10 μm spatial resolution.

Statistical analysis
The relationship between the LILRB4 expression and bone lesion was analyzed by the linear regression in GraphPad Prism 10.0 software.One-way ANOVA or t test was used to analyses the differences among the different groups.
Next, we collected 9 fresh specimens of multiple myeloma from UT Southwestern Medical Center and detected LILRB4 level by flow cytometry.CD45 − /CD38 + cells were malignant multiple myeloma cells [49].LILRB4 was detected in 55.56% multiple myeloma samples (5/9, Fig. 1B and Supplementary Fig. S2A).Moreover, LILRB4 was also expressed in multiple myeloma cell lines KMS26 and OPM2.LILRB4 was knocked out by CRISPR-cas9 technology in KMS26 and OPM2 cells (Fig. 1C).Meanwhile, the knockout of LILRB4 did not alter the proliferation of KMS26 and OPM2 cells (Supplementary Fig. S2B,  C), indicating that LILRB4 did not regulate the proliferation of multiple myeloma.
Extensive osteolytic bone lesion is the most characteristic feature of multiple myeloma.Although LILRB4 expressed on the surface of osteoclast negatively regulated osteoclastogenesis [43,52], the role of LILRB4 on bone lesion of multiple myeloma has not been reported.We collected bone marrow specimen and imaging data of 16 newly diagnosed patients with multiple myeloma, and found that the higher the expression level of LILRB4, the more serious the bone lesion (Fig. 1D and Supplementary Fig. S2D), suggesting that there was a certain trend and LILRB4 may be involved in bone injury in multiple myeloma.

The conditioned medium from LILRB4-WT not -KO cells significantly promoted osteoclastogenesis
Osteoclast is the only cell that can promote bone resorption [53], and multiple myeloma cells promotes the differentiation and maturation of osteoclasts [54].It has not been reported whether LILRB4 on multiple myeloma cells is involved in osteoclastogenesis.The conditioned medium (CM) from LILRB4-WT and -KO cells was used to treat the mouse macrophage cell line Raw264.7.Compared to the PBS group, RANKL could induce the osteoclastogenesis (more than 3 nuclei, Supplementary Fig. S3A, B).The CM from KMS26 or OPM2 could significantly induce the differentiation and maturation of osteoclast, and the numbers of osteoclasts in LILRB4-WT group were significantly higher than that in LILRB4-KO group (Supplementary Fig. S3A, B).Next, human LILRB4 was overexpressed in mouse myeloma cell line J558, and the CM was used to treat the primary mouse bone marrow mononuclear cells.The CM from J558-LILRB4 cells could significantly induce osteoclastogenesis (Supplementary Fig. S3C, D) when compared with the CM from J558-vector cells, suggesting that LILRB4 on multiple myeloma cells could induce osteoclastogenesis.
To determine the effect of myeloma-expressing LILRB4 on osteoblasts, we employed the CM of LILRB4-WT or -KO cells to treat MC3T3-E1 cell line (mouse embryonic calvarial fibroblasts).Alizarin red and ALP staining were used to characterize the maturation of osteoblasts.After 14 days of CM treatment, the cellular boundary was unclear, the morphology disappeared, and the apoptosis was obvious, indicating the CM from multiple myeloma, whether LILRB4 was knocked out or not, could induce the apoptosis of osteoblasts (Supplementary Fig. S4A-D).There was no significant difference between these two groups by alizarin red and ALP staining (Supplementary Fig. S4A-D), indicating the knockout of LILRB4 did not improve the osteoblasts apoptosis mediated by multiple myeloma, and also did not promote the differentiation and maturation of osteoblasts.
These results suggested that LILRB4 on multiple myeloma promoted the osteoclastogenesis, and had no effect on the differentiation and maturation of osteoblasts.

LILRB4 promoted osteolytic lesions in vivo
Next, we used different animal models [55] to study whether LILRB4 in multiple myeloma can mediate osteolytic lesions.KMS26-LILRB4-WT and -KO cells were injected into NSG mice by tail vein (Fig. 2A).After 35 days, the mice showed detectable somatic and nervous system symptoms, such as paraplegia of the hind limbs, fixed lap running, and all mice were sacrificed under anesthesia.Bioluminescence imaging and the numbers of tumor in abdominal cavity showed no significant difference in proliferation of KMS26-LILRB4-WT and-KO cells in vivo (Supplementary Fig. S5A-D).The 3D reconstruction of tibia showed that five of the seven mice had obvious bone fractures and bone lesions in KMS26-LILRB4-WT group, however, the tibia of all mice in KMS26-LILRB4-KO group were intact, and there was no visible bone damage (Fig. 2B).Trabecular bones were almost completely destroyed, bone cortex was discontinuum, and 3D-trabecular had obvious defects in LILRB4-WT not -KO group (Fig. 2C, D).Micro-CT analysis of tibial trabecular bone volume / tissue volume (BV/TV), trabecular bone surface / tissue volume (BS/ TV) and bone mineral density (BMD) were significantly lower in LILRB4-WT group than that in LILRB4-KO group (Fig. 2E-G).Histological analysis demonstrated that cortical and trabecular bone had obvious lesions in KMS26-LILRB4-WT not -KO group (Fig. 2H).In OPM2 xenograft models, 4 out of 5 mice (80%) showed significant cracking in their cranial sutures in LILRB4-WT group, but only 2 mice (40%) showed crack in LILRB4-KO group (Supplementary Fig. S6A), and the knockout of LILRB4 could significantly prolong the survival time of mice (Supplementary Fig. S6B).LILRB4 significantly promoted bone lesion, and trabecular destruction was more serious (Supplementary Fig. S6C, D).BV/TV, BS/TV and BMD also showed that LILRB4-WT not -KO group had stronger bone destruction (Supplementary Fig. S6E-G).There is no significant difference in the proportion of tumor cells in peripheral blood (Supplementary Fig. S6H).The above results showed that LILRB4 did not promote the proliferation of multiple myeloma, and the bone lesion mediated by LILRB4 was not caused by the proliferation of multiple myeloma cells.
To further confirm the above results, we used 5TGM1 and patient derived xenograft (PDX) models to observe the bone lesions mediated by LILRB4.Human LILRB4 was expressed in 5TGM1 cells (Fig. 3A), and 5TGM1vector and -LILRB4 cells were injected into C57BL/6-Rag2 −/− mice [56].Bioluminescence imaging showed that there was no significant difference between 5TGM1vector and -LILRB4 groups (Supplementary Fig. S7A, B), indicating that LILRB4 did not promote the cell proliferation.The bone damage of tibia was obvious in 5TGM1-LILRB4 group (Fig. 3B), and trabecular bone damage was more profound (Fig. 3C, D), and BV/TV, BS/TV were lower in 5TGM1-LILRB4 group (Fig. 3E, F).Histological staining showed that the cortical and trabecular bone was more intact in 5TGM1-vector group than that in -LILRB4 group (Supplementary Fig. S7C).Next, fresh bone marrow samples of multiple myeloma were collected.CD45 − /CD38 + /LILRB4 + and CD45 − /CD38 + /LILRB4 − myeloma cells from the same patient were sorted, and the same number of cells were injected into NSG mice by intratibial injection, respectively.When mice died naturally, the bone lesion was scanned by micro-CT.CD45 − / CD38 + /LILRB4 + cells significantly promoted the bone damage (Fig. 3G), and trabecular bone damage was more serious in CD45 − /CD38 + /LILRB4 + group than that in CD45 − /CD38 + /LILRB4 − group (Fig. 3H-L).Collectively, ectopic LILRB4 significantly promoted bone lesion of multiple myeloma.

RELT is upregulated in LILRB4-WT cells of multiple myeloma cell lines
To clarify the mechanism by which LILRB4 promotes bone lesion of multiple myeloma, we used cytokine arrays to identify the differentially expressed cytokines in the CM from LILRB4-WT and -KO cells.Compared with LILRB4-KO group, 380 and 299 cytokines were upregulated in KMS26 and OPM2-LILRB4-WT cells, respectively.The upregulated cytokines were enriched in multiple pathways including Ras, PI3K-Akt, NF-κB, MAPK, JAK-STAT, IL-17 and cytokine-cytokine receptor interaction (Fig. 4A, B).VEGF, RANKL, RELT, TRPC1 and P53 are related with osteolysis [57].Real-time PCR results indicated that RELT and P53 were significantly lower in LILRB4-KO than that in LILRB4-WT cells of KMS26 and OPM2 (Fig. 4C).Meanwhile, RNA-seq was used to screen the differentially expressed genes, and a total of 5496 differential genes were found, in which 835 genes were co-upregulated in LILRB4-WT cells (Supplementary Fig. S8A).These co-upregulated genes were enriched in the following pathways including cytokinecytokine receptor interaction, chemokine signaling pathway, JAK-STAT and NF-κB signal pathway (Supplementary Fig. S8B), and RELT, IL27RA, IL4R, TGFB1 and TNFRSF1B were significantly higher in LILRB4-WT group than that in LILRB4-KO group (Supplementary Fig. S8C, D).
Based on the above results, we speculated that LILRB4 could promote the secretion of RELT.We detected the RELT level in CM from KMS26 and OPM2, and found that RELT was higher in LILRB4-WT group than that in LILRB4-KO group (Fig. 4D).Next, we constructed the dual luciferase reporter system with RELT promoter, and this system was transfected into the LILRB4-WT and -KO cells.The relative luciferase activity (RLA) could hardly be detected in LILRB4-KO cells, but the RLA was higher in LILRB4-WT cells (Fig. 4E).Meanwhile, LILRB4 plasmid and luciferase reporter system were co-transfected into OPM2-LILRB4-KO cells, and the co-transfection of LILRB4 could also promote the activity of dual luciferase reporter system (Fig. 4F).We have previously reported that LILRB4 activated the NF-κB signal pathway by recruiting p-SHP2 [31,32].Next, we confirmed that LILRB4 could recruit p-SHP2 in multiple myeloma cells by Co-IP (Fig. 4G), and the co-transfection of p65 could significantly promote the activity of dual luciferase reporter system (Fig. 4H).LILRB4 and p65 could both promote the expression of RELT.Western blotting showed that, compared to LILRB4-KO group, p-SHP2 and RELT were significantly higher in cytoplasm, and p-p65 was also significantly elevated in the nucleus in LILRB4-WT group (Fig. 4I and Supplementary Fig. S9A).
Taking together, these studies indicated that LILRB4 could recruit p-SHP2 to active NF-κB signal pathway to promote the secretion of RELT.

RELT induced osteoclastogenesis and bone lesions in MM models
Whether RELT promotes osteolytic lesions in multiple myeloma has not been reported.RELT was overexpressed in LILRB4-KO cells (named LILRB4-KO-RELT), and the RELT level in the CM from LILRB4-KO-RELT cells was significantly higher than that in LILRB4-KO-vector cells (Fig. 5A, B).Next, the CM from LILRB4-KO-RELT or -vector cells were used to treat primary bone marrow monocytes, and the CM of LILRB4-KO-RELT cells significantly promoted the osteoclastogenesis (Fig. 5C).Meanwhile, the similar phenotype was observed, that is, the recombinant human RELT could induce the differentiation and maturation of osteoclasts (Supplementary Fig. 5 RELT regenerated bone damage in LILRB4-KO cells.RELT was overexpressed in KMS26-LILRB4-KO cells (A), and the content of RELT in conditioned medium was determined by ELISA (n = 3, B).The conditioned medium from KMS26-LILRB4-KO-vector or -RELT was used to treat primary bone marrow monocytes (n = 3, C).3D structure of tibia (D), 3D trabecular (E), BV/TV and BS/TV (F) were analyzed by micro-CT (n = 18).RELT signal pathway was detected by western blotting (G) Fig. S9B).Next, KMS26-LILRB4-WT, KMS26-LILRB4-KO-RELT and -vector cells were injected into NSG mice by intratibial injection.After 7 days, mice were sacrificed under anesthesia, and micro-CT was used to scan the bone lesion.3D structure of tibia showed that the surface of bone cortex was uneven, and multiple obvious bone injures were observed, and this phenotype was more severe in LILRB4-WT group as well as LILRB4-KO-RELT group (Fig. 5D).3D-trabecular showed that trabecular loss was more severe, BV/TV and BS/TV was lower in LILRB4-WT and LILRB4-KO-RELT group (Fig. 5E-F).These results indicated that RELT overexpression in LILRB4-KO cells could completely restore the bone damage caused by LILRB4, then RELT does play an important role in sustaining the LILRB4 osteolytic function.
Next, we collected bone marrow samples from 5 cases of multiple myeloma, and found that higher level of LILRB4 in CD45 − /CD38 + multiple myeloma cells seems to correspond to a tendency towards more RELT level in serum (Supplementary Fig. S9C, D).RELT could promote the proliferation of T cells [58], however, it is unknown how RELT regulates osteoclastogenesis.The CM of KMS26-LILRB4-KO-vector or -RELT cells was used to treat Raw264.7 cells.After 4 days, the cells were lysed, and cytoplasmic and nuclear proteins were extracted, respectively.In the CM-RELT-treated group, the level of p-MEK was significantly increased in cytoplasm, and p-ERK, p-c-fos and NFATC1 were also significantly elevated in the nucleus, but p-AKT and p-p65 were no significant changed (Fig. 5G and Supplementary Fig. S9E-J).p-c-fos and NFATC1 are markers of osteoclast maturation and differentiation.Therefore, RELT promoted the proliferation, maturation and differentiation of osteoclast by p-MEK/p-ERK/p-c-fos and NFATC1 signal pathway.

The knockout of LILRB4, anti-LILRB4 alone or in combination with BTZ could alleviate bone lesions
Bortezomib (BTZ) is a protease inhibitor, a first-line therapy for the treatment of multiple osteoma in the clinical [59].Little is known whether BTZ can inhibit bone damage in multiple myeloma with the depletion of LILRB4.KMS26-LILRB4-WT or -KO cells were injected into NSG mice by tail vein, and BTZ or PBS was used to treat xenograft models twice a week (Fig. 6A).After 33 days, all mice were sacrificed under anesthesia, and bone lesion was canned by micro-CT.Compared with LILRB4-KO group, LILRB4-WT cells significantly promoted the bone injury (Fig. 6B, C).After BTZ treatment, the destruction of trabecular was significantly reversed, especially in LILRB4-KO group (Fig. 6D, E), and BV/TV, BS/TV were significantly elevated (Fig. 6F-G).Tb.Th was also increased, although not significantly (Fig. 6H).
Next, we used anti-LILRB4 and BTZ to treat KMS26 mouse model (Fig. 7A).After 40 days of tumor injection, 3 out of 6 mice (50%) died in isotype and only BTZ treated groups, 1 mouse (16.67%) died in anti-LILRB4treated group, and no mice died in the combination therapy group (anti-LILRB4 + BTZ).In isotype or BTZ treated groups, 2 out of 6 mice (30%) showed significant cracking in their cranial sutures, and only 1 mouse showed cracking in anti-LILRB4 and combination therapy (Fig. 7B).The bone erosion, bone fracture and trabecular bone loss were more severe in isotype or BTZ treated groups than that in anti-LILRB4 or combination therapy groups (Fig. 7C-F).BV/TV and BS/TV were significantly increased in anti-LILRB4 and combination therapy groups (Fig. 7G, H).
The above results indicated that anti-LILRB4 alone or in combination with BTZ have the potential to inhibit bone damage in multiple myeloma.

Discussion
LILRB4 is a molecular marker of M4 and M5 acute myeloid leukemia (AML), which mediates the immune escape and extramedullary infiltrates of AML [31,32].LILRB4 is also expressed on the surface of tumor related macrophages, MDSC and other immune cells in the tumor microenvironment, forming a suppressive tumor microenvironment to promote tumor cell immune escape, and is a potential target for immunotherapy [35,38,60].In addition, LILRB4 has been reported to be expressed in non-small cell lung cancer [42], therefore, elucidating the expression pattern of LILRB4 in tumor cells is fundamental for investigating the role of LILRB4.The surface proteomic analysis of multiple myeloma identified LILRB4 as a potential immunotherapeutic target [61].In this study, we first focused on the expression of LILRB4 in various tumor tissue arrays.Unexpectedly, LILRB4 was expressed in 50% of multiple myeloma samples, and flow cytometry further validated this result.Although LILRB4 was also expressed in several other solid tumors, the level was lower, therefore, we focused on and explored the relationship between LILRB4 and multiple myeloma.
Multiple myeloma is typically characterized by multiple bone lesion.We analyzed the relationship between patient imaging data and LILRB4 expression, and found that the patients with high expression level of LILRB4 had more severe bone damage.In vitro data also showed that LILRB4 could promote the differentiation and maturation of osteoclasts, but had no significant effect on the differentiation and maturation of osteoblasts.Next, we used differential animal models to validate this result.In xenograft model, the knockout of LILRB4 in KMS26 and OPM2 could significantly inhibit the destruction of bone cortex and trabecular.In syngeneic model, 5TGM1 cells were injected into the C57BL/6-Rag2 −/− mice by tail vein.The overexpression of LILRB4 in 5TGM1 cells significantly promoted bone damage.A paper reported that LILRB4 mediated the migration of MM cell line (NCI-H929) [62].However, we did not observe any effect of LILRB4 on MM cell migration in differential animal models.Finally, PDX models also showed that CD45 − /CD38 + /LILRB4 + cells had stronger bone destruction.
LILRB4 breaks the balance between osteoclasts and osteoblasts, resulting in more severe bone damage in the multiple myeloma mouse model.Subsequently, we explored the mechanism of LILRB4 promoting bone injury in multiple myeloma.The results of cytokine microarray, RNA-Seq and real time-PCR showed that RELT was the most significant upregulated cytokine in LILRB4-WT group, and dual luciferase reporting system showed that LILRB4 could regulate the secretion of RELT by p-SHP2-NF-κB signal pathway (Fig. 8).RELT, a new member of the tumor necrosis factor receptor superfamily, is selectively expressed in hematopoietic tissues and activates transcription factor NF-κB [58], and negatively regulates the activity of T cells [63].Serum RELT (soluble) was elevated in patients with gastric cancer and breast cancer [64,65], especially in B-cell lymphomas, but RELT is an orphan receptor, and its ligand is unknown [66].How RELT promotes the differentiation and maturation of osteoclasts has not been reported.RELT binds to its putative receptor(s) on osteoclasts, and drivers the maturation of osteoclasts.In this study, our data confirmed that RELT promoted the proliferation of osteoclasts by p-MEK/p-ERK/p-cfos and NFATC1 signal pathway (Fig. 8).
Finally, we tried to explore the role of LILRB4 in treatment of multiple myeloma.The depletion of LILRB4 or anti-LILRB4 alone or in combination with BTZ could delay the progression of multiple myeloma, and significantly inhibit the bone damage (Fig. 8).LILRB4 did not affect the proliferation and metastasis of multiple myeloma, but LILRB4 blocking antibody could prevent the binding of LILRB4 to its ligands, therefore, anti-LILRB4 could block its signal pathway to inhibit the RELT secretion, then alleviate bone damage.Our data supported that LILRB4 signal pathway promoted the differentiation and mature of osteoclasts by secreting RELT, and LILRB4 had the potential to be a new target for the treatment of multiple myeloma.

Fig. 1
Fig. 1 LILRB4 was expressed in multiple myeloma.LILRB4 was detected by IHC in tissue array of multiple myeloma (the arrow indicated the LILRB4 expression in normal bone marrow, n = 34, A), by flow cytometry in fresh patient samples (n = 9, B).LILRB4 was knocked out by CRISPR-Cas9 in KMS26 and OPM2 cell lines (C).The imaging data showed the relationship between LILRB4 level and bone damage (n = 16, D)

Fig. 2
Fig. 2 LILRB4 promoted bone injury in KMS26 xenograft model.The schematic of KMS26 xenograft model (A).Micro-CT was used to scan the bone lesion after 35 days.3D reconstruction of tibia (B), transverse plane of trabecular (C) and 3D-trabecular (D) showed the tibial morphology and the structure of the trabecular.Trabecular bone volume/tissue volume (BV/TV, E), trabecular bone surface/tissue volume (BS/TV, F) and bone mineral density (BMD, G) were analyzed by micro-CT (n = 14).Representative bone lesions were observed in H&E staining (H)

Fig. 6
Fig. 6 BTZ was used to treat LILRB4-WT and -KO mouse models.The schematic of BTZ treated KMS26 xenograft model (n = 16, A). 3D structure of tibia, sagittal and transverse plane of trabecular and 3D trabecular in different group were shown (B-E).BV/TV, BS/TV and Tb.Th (trabecular thickness) were determined by micro-CT (n = 16, F-H)

Fig. 7
Fig. 7 Anti-LILRB4 alone or in combination with BTZ treated KMS26 mouse model.The schematic to show the treatment of anti-LILRB4 and BTZ (A).The crack of cranial sutures was scanned by micro-CT (B).The 3D-structure, sagittal plane and 3D-trabecular in tibia were scanned by micro-CT (n = 24, C-F), and BV/TV, BS/TV were analyzed (G, H)